Bottom Line:
Small. 1999.Cell Biol. 146:1033-1043).In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole.

ABSTRACTRecent studies have shown that the targeting of substrate adhesions by microtubules promotes adhesion site disassembly (Kaverina, I., O. Krylyshkina, and J.V. Small. 1999. J. Cell Biol. 146:1033-1043). It was accordingly suggested that microtubules serve to convey a signal to adhesion sites to modulate their turnover. Because microtubule motors would be the most likely candidates for effecting signal transmission, we have investigated the consequence of blocking microtubule motor activity on adhesion site dynamics. Using a function-blocking antibody as well as dynamitin overexpression, we found that a block in dynein-cargo interaction induced no change in adhesion site dynamics in Xenopus fibroblasts. In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole. Blockage of kinesin activity had no influence on either the ability of microtubules to target substrate adhesions or on microtubule polymerisation dynamics. We conclude that conventional kinesin is not required for the guidance of microtubules into substrate adhesions, but is required for the focal delivery of a component(s) that retards their growth or promotes their disassembly.

fig5: Control of block in kinesin motor activity by SUK-4 antibody. Figure shows living Xenopus fibroblasts in which mitochondria were marked with Rhodamine-123. The upper cell was injected with SUK-4 plus TAMRA dextran (diffuse background label in cytoplasm). Mitochondria are collapsed around the nucleus in the injected cell.

Mentions:
Control experiments showed that injection of the SUK-4 antibody consistently induced the collapse of mitochondria into the perinuclear area (Fig. 5) diagnostic of kinesin inhibition (Rodionov et al., 1993).We then investigated the influence of kinesin inhibition in cells injected with rhodamine-vinculin. As shown in Fig. 6, A and B , a block in kinesin activity by injection of SUK-4 Ab caused a dramatic change in the pattern of substrate adhesions over the 3-h assay period. The change observed was similar to that induced by microtubule disassembly with nocodazole (Fig. 1), and characteristically involved a major lengthening and reduction in the number of adhesion sites (Fig. 2). Similar changes after SUK-4 antibody injection were observed in time-lapse videos of cells transfected with GFP-zyxin (Fig. 6, C and D; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200105051.DC1). Because zyxin also binds to stress fiber assemblies, the growth and lengthening of adhesions could be readily correlated with the growth of stress fiber bundles (Fig. 6, C and D).

fig5: Control of block in kinesin motor activity by SUK-4 antibody. Figure shows living Xenopus fibroblasts in which mitochondria were marked with Rhodamine-123. The upper cell was injected with SUK-4 plus TAMRA dextran (diffuse background label in cytoplasm). Mitochondria are collapsed around the nucleus in the injected cell.

Mentions:
Control experiments showed that injection of the SUK-4 antibody consistently induced the collapse of mitochondria into the perinuclear area (Fig. 5) diagnostic of kinesin inhibition (Rodionov et al., 1993).We then investigated the influence of kinesin inhibition in cells injected with rhodamine-vinculin. As shown in Fig. 6, A and B , a block in kinesin activity by injection of SUK-4 Ab caused a dramatic change in the pattern of substrate adhesions over the 3-h assay period. The change observed was similar to that induced by microtubule disassembly with nocodazole (Fig. 1), and characteristically involved a major lengthening and reduction in the number of adhesion sites (Fig. 2). Similar changes after SUK-4 antibody injection were observed in time-lapse videos of cells transfected with GFP-zyxin (Fig. 6, C and D; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200105051.DC1). Because zyxin also binds to stress fiber assemblies, the growth and lengthening of adhesions could be readily correlated with the growth of stress fiber bundles (Fig. 6, C and D).

Bottom Line:
Small. 1999.Cell Biol. 146:1033-1043).In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole.

ABSTRACTRecent studies have shown that the targeting of substrate adhesions by microtubules promotes adhesion site disassembly (Kaverina, I., O. Krylyshkina, and J.V. Small. 1999. J. Cell Biol. 146:1033-1043). It was accordingly suggested that microtubules serve to convey a signal to adhesion sites to modulate their turnover. Because microtubule motors would be the most likely candidates for effecting signal transmission, we have investigated the consequence of blocking microtubule motor activity on adhesion site dynamics. Using a function-blocking antibody as well as dynamitin overexpression, we found that a block in dynein-cargo interaction induced no change in adhesion site dynamics in Xenopus fibroblasts. In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole. Blockage of kinesin activity had no influence on either the ability of microtubules to target substrate adhesions or on microtubule polymerisation dynamics. We conclude that conventional kinesin is not required for the guidance of microtubules into substrate adhesions, but is required for the focal delivery of a component(s) that retards their growth or promotes their disassembly.